Golgi proteomics : Identification of a novel cartilage-specific Golgi protein GoPro49

The Golgi complex is a central organelle of the secretory pathway, responsible for a range of post-translational modifications, as well as for membrane traffic to the plasma membrane and to the endosomal-lysosomal pathway. In addition, this organelle has roles in cell migration, in the regulation of traffic, and as a mitotic check point. The structure of the Golgi complex is highly dynamic and able to respond to the amount of cargo being transported and the stage of the cell cycle. The Golgi proteome reflects the functions and structure of this organelle, and can be divided into three major groups: the Golgi resident proteins (e.g. modification enzymes), the Golgi matrix proteins (involved in structure and tethering events), and trafficking proteins (e.g. vesicle coat proteins and Rabs). The Golgi proteome has been studied on several occasions, from both rat liver and mammary gland Golgi membranes using proteomic approaches, but still little more than half of the estimated Golgi proteome is known. Nevertheless, methodological improvements and introduction of shotgun proteomics have increased the number of identified proteins, and especially the number of identified transmembrane proteins. Cartilage, even though not a typical tissue in which to study membrane traffic, secretes large amounts of extracellular matrix proteins that are extensively modified, especially by amino acid hydroxylation, glycosylation and sulfation. Furthermore, the cartilage ECM contains several, large oligomeric proteins (such as collagen II) that are difficult to assemble and transport. Indeed, cartilage has been shown to be susceptible to changes both in secretory pathway (e.g. the COPII coat assembly) and in post-translational modifications (e.g. heparan sulfate formation). Dental follicle, and the periodontal ligament (PDL) that it forms, are another type of connective tissue, and they have a role in anchoring teeth to bone. This anchorage is achieved by numerous matrix fibres that connect the bone matrix with the cementum. These tissues have in common the secretion of large matrix molecules. In this study the Golgi proteome was analysed from purified, stacked Golgi membranes isolated from rat liver. The identified, extensive proteome included a protein similar to Ab2-095, or Golgi protein 49kDa (GoPro49), which was shown to localise to the Golgi complex as an EGFP fusion protein. Surprisingly, in situ hybridisation showed the GoPro49 expression to be highly restricted to different mesenchymal tissues, especially in cartilage, and this expression pattern was clearly developmentally regulated. In addition to cartilage, GoPro49 was also expressed in the dental follicle, but was not observed in the mature PDL. Importantly, GoPro49 is the first specific marker for the dental follicle. Endogenous GoPro49 protein co-localised with β-COP in both chondrosarcoma and primary dental follicle cell lines. The COPI staining in these cells was highly dynamic, showing a number of tubules. This may reflect the type of secretory cargo they secrete. Currently GoPro49 is the only Golgi protein with such a restricted expression pattern.Jokaisessa solussamme on useita erilaisia soluelimiä suorittamassa solulle välttämättömiä tehtäviä. Yksi niistä on Golgin laite, joka sijaitsee tuman vieressä ja vastaa proteiinin kuljetuksesta solukalvolle ja ulos solusta. Golgin laite vastaa myös eritettävien proteiinien muokkauksesta entsymaattisesti, liittämällä esimerkiksi sokeriketjuja, rasvahappoja tai muita proteiinien toimintaan vaikuttavia ryhmiä proteiinin aminohappoihin. Jotta Golgin laite pystyisi toimittamaan kaikki tehtävänsä, sillä on joukko omia proteiineja, eli Golgin proteomi, vastaamassa näistä tehtävistä. Golgin proteomia on tutkittu useaan otteeseen, mutta edellen osa siihen kuuluvista proteiineista on tunnistamatta. Monet elimistömme kudokset tuottavat ja erittävät runsaasti proteiineja, kuten esimerkiksi maksa albumiinia ja hyytymistekijöitä verenkiertoon. Myös rusto, joka muodostaa tukirangan yksilönkehityksen aikana, erittää aktiivisesti proteiineja soluvälitilaan. Nämä sidekudoksen proteiinit ovat tärkeitä ruston toiminnalle, esimerkiksi sen kestävyydelle, ja muodostavat merkittävän osan tästä kudoksesta. Tärkeää tälle sidekudoksen osalle ja koko ruston toiminnalle on myös proteiinien muokkaus ja mm. sokeriryhmien liittäminen. Tämän vuoksi Golgin laitteen toiminta ja proteiinien kuljetus ovat tärkeitä ruston toimin kannalta. Väitöskirjatyössäni olen tutkinut Golgin proteomia ja olemme tunnistaneet suuren joukon uusia tuntemattomia proteiineja. Näistä proteiineista yksi valittiin tarkemman tutkimuksen kohteeksi. Näytämme, että tämä proteiini, jonka nimesimme Golgi proteiini 49kDa:ksi (GoPro49), on uusi, Golgin laitteessa esiintyvä proteiini, jonka sijainti solussa on yhdenmukainen beta-COP proteiinin kanssa. Beta-COPia tarvitaan proteiinien kuljettamiseen Golgin laitteseen ja sieltä pois. Tämä havainto saattaa tarkoittaa, että GoPro49 proteiinin tehtävä solussa liittyy proteiinien kuljetukseen. Yllätykseksemme totesimme, että GoPro49 ilmentyy hiiren yksilönkehityksen aikana pääosin rustossa ja hampaita ympäröivässä sidekudoksessa. Tämä on epätavallista Golgin proteiinille, koska Golgin laite on välttämätön kaikille soluille ja siksi Golgin proteiineja on yleensä kaikissa soluissamme. Tämä saattaa kertoa, että proteiinia tarvitaan eritysesti tiettyjen, vain sidekudoksessa esiintyvien proteiinen kuljetukseen, tai että ruston proteiinit vaativat tavallista enemmän apua kuljetuksessa. Tuloksiemme perusteella GoPro49 on mielenkiintoinen uusi proteiini. Sen tarkka tehtävä rusto-solussa vaatii kuitenkin vielä tarkempaa tutkimusta

Extracellular vesicles from human plasma and serum are carriers of extravesicular cargo-Implications for biomarker discovery

Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.Peer reviewe

Metastatic state of parent cells influences the uptake and functionality of prostate cancer cell-derived extracellular vesicles

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Regulation of Calvarial Osteogenesis by Concomitant De-repression of GLI3 and Activation of IHH Targets

Loss-of-function mutations in GLI3 and IHH cause craniosynostois and reduced osteogeneiss, respectively. In this study, we show that ihh ligand, the receptor Ptch1 and Gli transcription factors are differentialyy expressed in embryonic mouse calvaria osteogenic condenstions. We show that in both ihh(-/-) and Gli3(Xt-J/Xt-J) embroyonic mice, the normal gene expression architecture is lost and this results in disorganized calvarial bone developement. RUNX2 is a master regulatory transciption factor controlling osteogenesis. In the absence of Gli3, RUNX2 isoform II and IHH are upregulated, and RUNX2 isoform I downregulated. This is consistent with the expandeed and aberant osteogenesis observed in Gli3Xt-J/Xt-J mice, and consistent RunX2-t expression by relatively immature osteoprogenitors. ihh-/- mice exhibited small calvarial bones HH target genes, Ptch1 and Gli1, were absent. This indicates that IHH is the functional HH ligand, and that it is not compensated by another HH ligand. To decipher the roles and potential interaction of Gli3 and ihh. we generated ihh-/-; gli3Xt-J/Xt-J compound mutant mice. Even in the absence of ihh, Gli3 deletion was sufficient to induce aberrant precocious ossification across the developing suture, indicating that the carniosyostosis pehnotype of Gli3Xt-J/Xt-J mice is not dependent on IHH ligand. Also we found that ihh was not required for Runx2 expression as the expression of RUNX2 target genes was unaffected by deletion of Ihh. To test whether RUNX2 has a role upstream of IHH we performed RUNX2 siRNA knock down experiements in WT calvarial osteoblasts and explants and found that Ihh expression is suppressed. Our results show that IHH is the functional HH ligand in the embroynic mouse calvaria osteogenic condensations, where it regulates the progression of osteoblastic differentation. As GLI3 represses the expression of Runx2-II abd Ihh, and also elevats the Runx2-I expression, and as IHH may be regulated by RUNX2 these results raise the possibility of a regualtory feedback circuit to control calvarial osteogenesis and suture patency. Taken together RUNX2-controlled osteoblastic cell fate is regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstreams targets.Peer reviewe

Loss-of-function of Gli3 in mice causes abnormal frontal bone morphology and premature synostosis of the interfrontal suture

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Noggin null allele mice exhibit a microform of holoprosencephaly

Holoprosencephaly (HPE) is a heterogeneous craniofacial and neural developmental anomaly characterized in its most severe form by the failure of the forebrain to divide. In humans, HPE is associated with disruption of Sonic hedgehog and Nodal signaling pathways, but the role of other signaling pathways has not yet been determined. In this study, we analyzed mice which, due to the lack of the Bmp antagonist Noggin, exhibit elevated Bmp signaling. Noggin−/− mice exhibited a solitary median maxillary incisor that developed from a single dental placode, early midfacial narrowing as well as abnormalities in the developing hyoid bone, pituitary gland and vomeronasal organ. In Noggin−/− mice, the expression domains of Shh, as well as the Shh target genes Ptch1 and Gli1, were reduced in the frontonasal region at key stages of early facial development. Using E10.5 facial cultures, we show that excessive BMP4 results in reduced Fgf8 and Ptch1 expression. These data suggest that increased Bmp signaling in Noggin−/− mice results in downregulation of the hedgehog pathway at a critical stage when the midline craniofacial structures are developing, which leads to a phenotype consistent with a microform of HP

Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi) RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls. Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV-and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data. Results: Approximately 1 x 10(10) EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p <0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples. Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials.Peer reviewe

RAB23 regulates musculoskeletal development and patterning

RAB23 is a small GTPase which functions at the plasma membrane to regulate growth factor signaling. Mutations in RAB23 cause Carpenter syndrome, a condition that affects normal organogenesis and patterning. In this study, we investigate the role of RAB23 in musculoskeletal development and show that it is required for patella bone formation and for the maintenance of tendon progenitors. The patella is the largest sesamoid bone in mammals and plays a critical role during movement by providing structural and mechanical support to the knee. Rab23 ( -/- ) mice fail to form a patella and normal knee joint. The patella is formed from Sox9 and scleraxis (Scx) double-positive chondroprogenitor cells. We show that RAB23 is required for the specification of SOX9 and scleraxis double-positive patella chondroprogenitors during the formation of patella anlagen and the subsequent establishment of patellofemoral joint. We find that scleraxis and SOX9 expression are disrupted in Rab23 ( -/- ) mice, and as a result, development of the quadriceps tendons, cruciate ligaments, patella tendons, and entheses is either abnormal or lost. TGF beta-BMP signaling is known to regulate patella initiation and patella progenitor differentiation and growth. We find that the expression of TGF beta R2, BMPR1, BMP4, and pSmad are barely detectable in the future patella site and in the rudimentary tendons and ligaments around the patellofemoral joint in Rab23 ( -/- ) mice. Also, we show that GLI1, SOX9, and scleraxis, which regulate entheses establishment and maturation, are weakly expressed in Rab23 ( -/- ) mice. Further analysis of the skeletal phenotype of Rab23 ( -/- ) mice showed a close resemblance to that of Tgf beta 2 ( -/- ) mice, highlighting a possible role for RAB23 in regulating TGF beta superfamily signaling.Peer reviewe